WPI pHOptica Micro User manual

pHOpticaTM micro

Fiber Optic pH System

for pH microsensors

INSTRUCTION MANUAL

PC-controlled one-channel fiber optic pH system for pH

microsensors; excitation wavelength of 470nm; quartz-

quartz glass-fibers of 140 μm outer diameter connected

by ST-fiber connectors;

www.wpiinc.com

Table of Contents

1 Preface.............................................................................................................................1

2 Safety Guidelines ...........................................................................................................2

3 Description of the pHOptica micro Device...................................................................4

4 Required Basic Equipment............................................................................................7

5 pH-Sensitive Microsensors: Sensors and Housings ..................................................8

5.1 Housings of pH-Sensitive Microsensors............................................................11

5.1.1 Needle-type - pH Micro-Sensor (NTH)............................................................12

5.1.2 Implantable pH-Micro-Sensor .........................................................................14

6 Description of pHOptica micro Software ...................................................................17

6.1 Software Installation and Starting the Instrument.............................................17

6.2 Function and Description of the pHOptica micro Program..............................18

6.2.1 Menu Bar ........................................................................................................19

6.2.2 Control Bar......................................................................................................24

6.2.3 Graphical Window...........................................................................................30

6.2.4 Status Bar .......................................................................................................31

6.3 Subsequent Data Handling..................................................................................31

7 Calibration and Measurement.....................................................................................33

7.1 Buffers for Calibration..........................................................................................33

7.2 Mounting the Implantable Microsensors............................................................33

7.3 Mounting the Needle-Type Microsensors ..........................................................34

7.4 pHSolver-v07.exe used for calculation of the calibration values.....................37

7.5 Calibration/Measurement of a pH Microsensors...............................................39

7.6 Some Advice for Correct Measurement .............................................................40

7.6.1 Signal drift due to photo-decomposition .........................................................40

7.6.2 Performance proof ..........................................................................................41

8 General Instructions: ...................................................................................................42

8.1 Warm-Up Time ......................................................................................................42

8.2 Maintenance..........................................................................................................42

9 Technical Data..............................................................................................................43

9.1 General Data..........................................................................................................43

9.2 Analog Output and External Trigger...................................................................45

9.3 Technical Notes....................................................................................................46

9.4 Operation Notes....................................................................................................46

10 Appendix.......................................................................................................................47

10.1 Basics in Optical Sensing....................................................................................47

10.1.1 Major Components of Fiber-Optic Microsensors ............................................47

10.1.2 Luminescence Decay Time ............................................................................47

10.1.3 Advantages of Optical Lifetime Based Sensors..............................................48

10.1.4 Dynamic Quenching of Luminescence ...........................................................49

10.1.5 Dual Lifetime Referenced (DLR) Optical Sensors ..........................................50

Table of Contents

10.1.6 Fluorescence (Förster) Energy Transfer (FET) ..............................................52

10.1.7 Literature.........................................................................................................53

Preface

1

1 Preface

Congratulations!

You have chosen a new innovative technology to read out chemo-optical sensors!

The pHOptica micro is a compact, portable, PC-controlled fiber-optic phase detection device

to read out pH sensors. The data evaluation is PC supported.

The pHOptica micro was specially developed for very small fiber-optic microsensors. It is

based on a novel technology, which creates very stable, internal referenced measured

values. The pHOptica micro is useful to read out sensors with average luminescence decay

times in the range of 1 µs to 0.6 ms.

Optical sensors (also called optodes) based on decay time measurements have important

advantages over intensity based optodes:

• They are small

• They are independent on fluctuations of the light source

• Their signal does not depend on the flow rate of the sample

• They are less sensitive to photo bleaching and dye leaching

• They are not influenced by intensity fluctuations due to fiber bending

Therefore, they are ideally suited for the examination of small sample volumes, long term

measurements in difficult samples, and for biotechnological applications.

A set of different microsensors, flow through cells and integrated sensor systems is available

to make sure you have the sensor which is ideally suited to your application.

Please feel free to contact our service team to find the best solution for your application.

Safety Guidelines

2

2 Safety Guidelines

PLEASE READ THESE INSTRUCTIONS CAREFULLY BEFORE WORKING WITH THIS

INSTRUMENT!

This device has left our works after careful testing of all functions and safety requirements.

The perfect functioning and operational safety of the instrument can only be ensured if the

user observes the usual safety precautions as well as the specific safety guidelines stated in

these operating guidelines.

- Before connecting the device to the electrical supply network, please ensure that the

operating voltage stated on the power supply corresponds to the mains voltage

- The perfect functioning and operational safety of the instrument can only be

maintained under the climatic conditions specified in Chapter 9 "Technical Data" in

this operating manual.

- If the instrument is moved from cold to warm surroundings, condense may form and

interfere with the functioning of the instrument. In this event, wait until the

temperature of the instrument reaches room temperature before putting the

instrument back into operation.

- Balancing, maintenance and repair work must only be carried out by a suitable

qualified technician, trained by us.

- Especially in the case of any damage to current-carrying parts, such as the power

supply cable or the power supply itself, the device must be taken out of operation and

protected against being put back into operation.

- If there is any reason to assume that the instrument can no longer be employed

without a risk, it must be set aside and appropriately marked to prevent further use.

- The safety of the user may be endangered, e. g., if the instrument

• is visibly damaged;

• no longer operates as specified;

• has been stored under adverse conditions for a lengthy period of time;

• has been damaged in transport

- If you are in doubt, the instrument should be sent back to the manufacturer WPI for

repair and maintenance.

- The operator of this measuring instrument must ensure that the following laws and

guidelines are observed when using dangerous substances:

• EEC directives for protective labor legislation;

• National protective labor legislation;

• Safety regulations for accident prevention;

• Safety data-sheets of the chemical manufacturer

The pHOptica micro is not protected against water spray;

The pHOptica micro is not water proof;

The pHOptica micro must not be used under environmental conditions which cause water-

condensation in the housing;

The pHOptica micro is sealed;

The pHOptica micro must not be opened;

We explicitly draw your attention to the fact that any damage of the manufactural seal will

render of all guarantee warranties invalid.

Safety Guidelines

3

Any internal operations on the unit must be carried out by personal explicitly authorized by

WPI and under antistatic conditions.

Needle-type sensors are housed in extremely sharp syringe needles. Avoid injury by

handling the needle carefully. Please pay attention to all safety guidelines for safe handling

of sharp needles and syringes. Beware of injuring with the needle as well as with the sensor

tip. The glass fiber can break if pricked into the skin and can cause inflammation.

The pHOptica micro may only be operated by qualified personal.

This measuring instrument was developed for use in the laboratory. Thus, we must assume

that, as a result of their professional training and experience, the operators will know the

necessary safety precautions to take when handling chemicals.

Keep the pHOptica micro and the equipment such as PT1000 temperature sensor, power

supply and sensors out of the reach of children!

As the manufacturer of the pHOptica micro, we only consider ourselves responsible for

safety and performance of the device if

• the device is strictly used according to the instruction manual and the safety guidelines

• the electrical installation of the respective room corresponds to the DIN IEC/VDE

standards.

The pHOptica micro and the sensors must not be used in vivo examinations on humans!

The pHOptica micro and the sensors must not be used for human-diagnostic or

therapeutically purposes!

Description of the pHOptica micro Device

4

3 Description of the pHOptica micro Device

The pHOptica micro is a precise single channel

Fiber Optic pH System for pH microsensors based

on quartz-quartz glass-fibers of 140 μm. The small

outer dimensions, low power consumption and a

robust box make it ready for portable and field use.

For operation, a PC/Notebook is required. The

pHOptica micro is controlled using a comfortable

software, which also saves and visualizes the

measured values.

The pHOptica micro has 2 analog outputs (0-5 V)

and one trigger input (TTL) to be connected to a

data logger. Analog connectors are BNC

connectors.

The analog outputs are programmable to deliver

pH, temperature, or the raw values (phase shift or

amplitude), and the data are called via computer

and RS232 (digital) or using the external trigger

input (analog).

pHOptica micro instruments features:

• high precision

• portable (battery power optional)

• analog/digital data output

• external temperature measurement

Extension to a multi-channel

multi-analyte system

Using a computer port extender providing multiple

RS232 ports, up to eight single devices (OXY MICRO,

OXY MINI, pHOptica micro, …) can be connected to

one single computer. This multi-instrument set-up offers

a highly flexible method to create multi-channel, multi-

analyte measuring systems including additional

temperature-compensation of each channel.

Description of the pHOptica micro Device

5

Front Panel

ELEMENT DESCRIPTION FUNCTION

POWER ON/OFF switch Switches the device ON and OFF

SENSOR ST fiber connector

Connect the fiber-optic sensor here.

L1 Control

LED

red: instrument off;

green: instrument on;

orange: stand by;

Temp Connector for PT 1000

temperature sensor

Connect the PT 1000 temperature sensor for

temperature compensated measurements here.

Description of the pHOptica micro Device

6

Rear Panel of the pHOptica micro device

Two standard BNC connectors are added for analog output channels 1 and 2, another one

for external trigger input.

The electrical specifications of all rear panel connectors are given in technical specification

sheet. Please read also the technical notes to avoid mistakes.

ELEMENT DESCRPTION FUNCTION

12 VCD Line adapter for power

supply

Connector for 9 - 36 V DC power supply.

RS 232 RS232 interface

(male)

Connect the device with a RS232 data cable to your

PC/Notebook here.

CH 1 Analog out

(channel 1)

Connect the device with external devices, e.g. a data

logger

CH 2 Analog out

(channel 2)

Connect the device with external devices, e.g. a data

logger

EXT TRIG External trigger input Connect the device with external devices, e.g. data

logger with a trigger output, pulse generator

Required Basic Equipment

7

4 Required Basic Equipment

• pH meter pHOptica micro*

• Software pHOpt_v1.exe for pHOptica micro*

• PC / Notebook

(System requirements:

Windows 95/98/2000/Millenium/NT 4.0/XP; Pentium processor, at least 133 MHz, 16 MB

RAM)

• RS 232 Cable *

• Line adapter (110 - 220 V AC, 12 V DC) *

• Temperature sensor PT 1000*

• Analyte-sensitive microsensor

The sensors are mounted into different types of housings

• Vessels for calibration solutions

We recommend Schott laboratory bottles with a thread which can be obtained by VWR International

(ordering number: 215L1515)

• Laboratory support with clamp, sensor-manipulator

*: scope of supply

Fiber-Optic Microsensors: Sensors and Housings

8

5 pH-Sensitive Microsensors: Sensors and Housings

Optical pH Sensors designed for pHOptica micro device are based on the new Dual

Luminophor Reference method (see also appendix). They consist of an inert long decay time

reference dye and a short decay time indicator dye, which changes its fluorescence intensity

due to the pH-value. The measured value, the average decay time, represents the ratio of

the two fluorescence intensities. Therefore, the signal is internal referenced and insensitive

to fluctuations of the light source.

Dynamic range

Optical pH sensors respond according to the mass acting law, hence, they do not show a

linear behavior like potentiometric pH electrodes. This limits their use to a dynamic range of

approximately 3 to 4 pH units. On the other hand, the resolution can be very high in the

optimal range. Figure 5.1 shows a typical response curve of a pH sensor. Increasing the pH,

the signal - in our case the phase angle Φ - decreases. The phase angle Φ can be related to

the pH as shown in Figure 5.2. The theoretical aspects are explained more detailed in the

appendix.

3

4

5

6

7

8

9

10

01234

pH

time, min

456789

15

20

25

30

35

40

45

50

55

60

Imax : 18.77

Imin : 56.33

dpH : 00.64

pH0 : 06.51

phase

pH

Figure 5.1 Response of pH micro-sensor pH-HP-5. Figure 5.2 Effect of the phase angle of a pH

microsensor (pH-HP-5) on different pH values.

The characteristic of a typical sensor are listed below:

Dynamic range: pH 5.5 to 8.5

Resolution: 0.01 pH

Drift due to bleaching: 0.03 per 1000 measuring points

Response time: less then 30 sec

Please note that the given values may differ from your special device and sensoric

application.

Fiber-Optic Microsensors: Sensors and Housings

9

Resolution

Figure 5.3. Resolution of the pH sensor can be up to 0.01 pH

Cross sensitivities

While pH electrodes are influenced by sulfide, electromagnetic fields or flow velocity the

optical pH measurement is interfered by ionic strength. This problem can be overcome by a

calibration with buffers of similar ionic strength than the sample.

456789

15

20

25

30

35

40

45

50

55

phase

pH

is50

is100

is200

Figure 5.4 Cross-sensitivity to ionic strength – we recommend to calibrate the sensor in pH-adjusted

media!

6.80

6.85

6.90

6.95

7.00

0.00 2.00 4.00 6.00 8.00 10.00

time, min

pH

Continous mode (measurement each second)

Fiber-Optic Microsensors: Sensors and Housings

10

Sensor drift

Like all optical chemo sensors, optical pH-sensors are suspect to photobleaching. Due to this

process the measured intensity (amplitude) is decreasing while the phase of the sensor is

increasing. This process is quite slow and depends on the used light intensity and the

duration of illumination. If you are in doubt whether a measured increase in phase is due to

photobleaching or decreasing pH of the sample, please lower the light intensity. If the drift is

not getting smaller, your signal drift is due to pH changes in your sample.

Figure 5.5 Drift due to bleaching: 0.03 per 1000 measuring points

Typical bleaching rates are listed below.

Sensor Drift due to Bleaching

1000 Measuring points 0.03 pH

1 hour in sec. Mode 0.1 pH

1 d in 1 min Mode 0.05 pH

Please notice, that bleaching rates also depend on the adjusted light intensity of the LED and the

individual instrument and sensor.

Temperature dependency

pH Micro sensors display, as any other pH sensors, a distinct temperature sensitivity. Some

typical calibration curves at different temperatures are shown below:

6

6.2

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

8

0 200 400 600 800 1000 1200 1400

Measuring points

pH

Fiber-Optic Microsensors: Sensors and Housings

11

45678910

15

20

25

30

35

40

45

50

55

Phase

pH

5 °C

15 °C

20 °C

30 °C

35 °C

40 °C

To obtain most reliable results we recommend to calibrate at the same temperature as used

for measurement. Higher temperature mimics lower pH. At pH 7 a deviation of about 0.08 ph

/ 5° C occurs.

Limitations

The offered pH sensors for pHOptica micro were specially designed for physiological

samples and media. Samples with extremely low ionic strength and low buffer content may

be not measured correct. To test whether the sample can be measured correct, try to

perform the calibration procedure with a buffer system which is as similar to the sample as

possible. If the calibration procedure does not result in a sigmoidal shaped calibration plot,

the system is not suited for the sensor. Please contact our service team for special sensors.

The measurement can also be influenced by small, highly fluorescent molecules like

fluorescein or rhodamin in the sample.

The sensors do not stand pH above 9 for prolonged time and organic solvents.

Response time

The response time (t90) of the pH sensor is dependent from the diffusion rate of protons

through the sensor layer, hence, the response time is dependent from the thickness of the

sensor layer and the stirring rate. The typical response (t90) time of a pH-micro sensor is

below 30 sec.

5.1 Housings of pH-Sensitive Microsensors

WPI fiber-optic pH microsensors are based on 140 µm silica optical fibers. To protect the

small glass fiber tip against breaking, suitable housings and tubings around it, depending on

the respective application, were designed.

WPI offers the following standard designs:

Fiber-Optic Microsensors: Sensors and Housings

12

5.1.1 Needle-type - pH Micro-Sensor (NTH)

Needle-type pH micro-sensors are miniaturized

fiber optic chemical sensors designed for all

research applications were a small tip size of ca.

140 µm are necessary. Needle-type pH micro-

sensors are ideal for pH profiling in sediment

and biofilms with a high spatial resolution. The

pH-sensitive tip of the optical fiber is protected

inside a stainless steel needle. Such a design is

the best for an easy penetration through a

septum rubber or any other harsh material as

well as secure for transportation. For

measurement purposes the sensor must be

pushed out from the needle.

Fiber-Optic Microsensors: Sensors and Housings

13

Schematic drawing of the NTH (Needle-type) micro-sensor

~

male fiber-optic plug

safety nut

spring

fiber cable

syringe housing

transport block

needle plastic base

glass fiber

sensor tip

syringe plunger

protective plastic cap

syringe needle

needle plastic base

syringe needle

FEATURES

• High spatial resolution (ca. 140 µm)

• Penetration probe for insertion into semisolids like sediments or biofilms

• Penetration through septa

• Easy to handle

• Different needle size available

• No reference electrode needed

• High temporal resolution

• Insensible towards electrical interferences and magnetic fields

SPECIFICATION

Tip size 140 µm

Measuring range 5 – 9 pH

Response time 30 sec

Resolution Up to 0.01 pH

Fiber-Optic Microsensors: Sensors and Housings

14

Drift

(per 1000 Measuring points) 0.03 pH

Temperature Range from 5 °C to + 50 °C

Cross-Sensitivity

Optical pH sensors display slight cross sensitivity to

ionic strength (salinity) and towards small

fluorescent molecules like dissolved indicator dyes.

ORDER

The NTH (Needle-type) pH sensors are offered with options to be specified in the purchase order

form. The order code key shown below (see also the example) defines the parameters. Please,

choose the parameters that best meet Your requirements.

NTH

NS

L

Needle-Type

Housing Analyte pH

Code of pH-

sensor coating Length of

Glass Fiber

- 2.5 m

- 5 m

- 10 m

Stainless Needle

Length [mm] / diameter [mm]

- 20 / 0.4

- 40 / 0.4

- 40 / 1.2

- 120 / 0.8

pH HP5

EXAMPLE

NTH

NS

L

pH HP5 540 0.4

With this code you will order a microsensor mounted in a needle-type housing (NTH), with the pH

sensitive coating HP5 with a glass fiber length of 5 m (L5) mounted in a stainless needle of 40 mm

length and 0.4 mm diameter (NS 40/0.4) .

5.1.2 Implantable pH-Micro-Sensor

Implantable pH micro-sensors are the

miniaturized fiber optic chemical sensor

designed for all research applications were a

small tip size of 140 µm are necessary.

Implantable pH micro-sensors are ideal for

costumer-specific set up. They were applied

for implantation in blood circuits and for

profiling with a high spatial resolution

Fiber-Optic Microsensors: Sensors and Housings

15

Schematic drawing of the Implantable micro-sensor

FEATURES

• High spatial resolution (ca. 140 µm)

• Easy to handle

• Most flexible for a wide range of applications

• High temporal resolution

• Insensible towards electrical interferences and magnetic fields

SPECIFICATION

Tip size 140 µm

Measuring range 5 – 9 pH

Response time 30 sec

Resolution Up to 0.01 pH

Drift

(per 1000 Measuring

points) 0.03 pH

Temperature Range from 5 °C to + 50 °C

Cross-Sensitivity

Optical pH sensors display

slight cross sensitivity to ionic

strength (salinity) and

towards small fluorescent

molecules like dissolved

indicator dyes.

Fiber-Optic Microsensors: Sensors and Housings

16

ORDER

The Implantable pH sensors are offered with options to be specified in the purchase order

form. The order code key shown below (see also the example) defines the parameters.

Please, choose the parameters that best meet Your requirements.

IMP

600/ 140/

900/

Implantable

Housing Analyte pH

Code of pH-

sensor coating

pH HP5

outer plastic cable

Diameter [µm] / length [m]

900 µm / 0 10

inner plastic cable

diameter [µm] / length [cm]

600 µm / 0 to customer request

length of bare glass fiber

Diameter [µm] / length [mm]

140 µm / 1 to customer request

EXAMPLE

IMP

600/ 140/

900/

pH HP5

512

This is the order code for the implantable micro-sensor (IMP) with the pH sensitive coating

HP5, with a fiber cable length of 5 m (900/5), a cable plastic jacket length of 1 cm (600/1), a

bare optical fiber length of 2 mm (140/2)

Description of pHOptica micro Software

17

6 Description of pHOptica micro Software

This software is compatible with Windows 95/98/2000/Millenium/NT4.0/XP.

6.1 Software Installation and Starting the Instrument

1. Insert the supplied disc/CD into the respective drive. Copy the file pHOpt_v1.exe onto

your hard disk. (for example, create C:\pHOptica_micro\ pHOpt_v1.exe). Additionally,

you may create a link (Icon) on your desktop.

2. Connect the pHOptica micro via the supplied serial cable to a serial port of your

computer. Tighten the cable with the screws on your computer and on the pHOptica

micro.

3. Connect the power supply.

4. Please close all other applications as they may interfere with the software. Start the

program pHOpt_v1.exe with a double click. The following information window appears:

5. If the right com port is adjusted this information window disappears within a few

seconds. If the wrong com port is adjusted you are asked to set the right com port:

With a right mouse click onto ‘com port’ you are able to set the right com port. Please

confirm your selection by clicking the ‘OK’ button. The information window disappears if

the right com port is adjusted.

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WPI pHOptica Micro User manual

WPI pHOptica Micro User manual

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